Mechanical loading of bone is important for maintenance of bone mass and structural stability of the skeleton. When bone is mechanically loaded, movement of fluid within the spaces surrounding bone cells generates fluid shear stress (FSS) that stimulates osteoblasts resulting in enhanced anabolic (bone forming) activity. The mechanisms by which osteoblasts convert the external stimulation of FSS into biochemical changes, a process known as mechanotransduction, remain poorly understood. Focal adhesions are prime candidates for transducing external stimuli. Focal adhesion kinase (FAK), a non-receptor tyrosine kinase found in focal adhesions, may play a key role in mechanotransduction. To examine the role of focal adhesion kinase in mechanotransduction, we used short interfering RNAs (siRNA) to target FAK expression in primary rat calvarial osteoblasts (RCOBs). Targeting of FAK expression will disrupt signaling through focal adhesions in RCOBs and allow us to study focal adhesion mechanotransduction. Once cells have been treated with the FAK siRNAs and exhibit decreased FAK expression, they will be exposed to varying periods of FSS or static culture conditions. The response to FSS will be evaluated by several different endpoints. One such endpoint is cyclooxygenase 2 (Cox-2) induction. Cox-2 is normally upregulated by SS stimulation, and we hypothesize that this induction is regulated by focal adhesion signaling through FAK. We hypothesize that knockdown of FAK will prevent FSS stimulated upregulation of Cox-2 expression, and we can determine this using western blot analysis. Another endpoint to be examined is osteoblast differentiation. Upon stimulation by FSS, osteoblasts exhibit an upregulation of physiologically relevent bone markers including osteopontin, osterix, bone sialoprotein, and core binding factor 1. Quantitative real-time PCR will be used to analyze the regulation of these bone markers upon treatment with siRNA and exposure to FSS and compared to the appropriate negative controls. These data will contribute to the overall understanding of bone metabolism. More specifically, these data will contribute to the bone anabolism in response to mechanical loading. It is our hope that these studies will provide information that may lead to new targets in bone therapy. [unreadable] [unreadable] [unreadable]